Induction of peripheral neuroblastomas in Syrian hamsters after injection as neonates with JC virus, a human polyoma virus.

Neuroblastomas developed in 10 of 31 Syrian hamsters inoculated intraocularly with JC virus, a human polyoma virus. The latent period was 6 to 11 months. Primary tumors occurred in the abdominal cavity, pelvis, mediastinum, and neck region. The origin of one tumor from the adrenal gland was demonstrated. Metastases were seen in the liver, bone marrow, and lymph nodes. Two neuroblastomas arising in this experiment were transplanted serially in weanling hamsters, and a tissue culture cell line was established from one of the transplanted tumors. T-antigen was detected in three of five primary tumors tested and in the transplanted tumors. Antibody against T-antigen was demonstrated in sera from five of six animals with neuroblastomas. Neuroblastomas also developed after combined s.c. and i.p. injection of JC virus.     

Effect of host age, virus dose, and route of inoculation on tumor incidence, latency, and morphology in Syrian hamsters inoculated intravenously with oncogenic DNA simian virus 40.

Three-week-old to 12-month-old male Syrian hamsters were inoculated iv with 10(8.5) median tissue culture infective dose of simian virus 40 (SV40). Three-week-old hamsters were similarly inoculated with aliquots of SV40 of progressively decreasing titers. The tumor incidence and, to a lesser extent, the tumor latency were directly dependent on the age of the animals at the time of virus exposure and on the dose of the virus. However, this age-dose dependence was not of the magnitude usually observed in hamsters inoculated with SV40 sc or im. Moreover, the wide morphologic spectrum of neoplasms induced, i.e., lymphosarcoma, reticulum cell sarcoma, and osteogenic sarcoma, by iv route of inoculation, contrasted sharply with the anaplastic and spindle-cell sarcomas which were the only types of malignant tumors resulting when other routes were used.     

GMCSF‐armed vaccinia virus induces an antitumor immune response

Oncolytic Western Reserve strain vaccinia virus selective for epidermal growth factor receptor pathway mutations and tumor‐associated hypermetabolism was armed with human granulocyte‐macrophage colony‐stimulating factor (GMCSF) and a tdTomato fluorophore. As the assessment of immunological responses to human transgenes is challenging in the most commonly used animal models, we used immunocompetent Syrian golden hamsters, known to be sensitive to human GMCSF and semipermissive to vaccinia virus. Efficacy was initially tested in vitro on various human and hamster cell lines and oncolytic potency of transgene‐carrying viruses was similar to unarmed virus. The hGMCSF‐encoding virus was able to completely eradicate subcutaneous pancreatic tumors in hamsters, and to fully protect the animals from subsequent rechallenge with the same tumor. Induction of specific antitumor immunity was also shown by ex vivo co‐culture experiments with hamster splenocytes. In addition, histological examination revealed increased infiltration of neutrophils and macrophages in GMCSF‐virus‐treated tumors. These findings help clarify the mechanism of action of GMCSF‐armed vaccinia viruses undergoing clinical trials.     

Ependymomas, malignant tumors of pancreatic islets, and osteosarcomas induced in hamsters by BK virus, a human papovavirus.

BK virus (BKV), a human papovavirus, was inoculated iv into 3-week-old Syrian golden hamsters. Between 2 1/2 and 9 months after inoculation, 82% of the animals developed tumors. The induced neoplasms were ependymoma, carcinoma of the pancreatic islets, osteosarcoma, adenocarcinoma, angiosarcoma, angioma, lymphoma, and seminoma. Hypersecretion of insulin, glucagon, C-peptide, and calcitonin was detected in tumors of pancreatic islets. BKV etiology of tumors was supported by the following evidence: 1) No tumors with BKV-specific markers appeared in animals given injections of buffer, animals inoculated with BKV neutralized by anti-BKV-specific serum, or uninoculated controls; 2) BKV tumor (T) antigen was detected by immunofluorescence and complement fixation tests in tumors of animals inoculated with infectious BKV and in transplanted tumors; 3) antibodies to BKV T-antigen were detected in sera of animals bearing primary or transplanted tumors; 4) BKV could be activated by Sendai virus-mediated fusion of neoplastic cells with susceptible Vero cells; and 5) no endogenous hamster oncornaviruses were found in tumors.     

Effects of the antifertility drug enovid in five strains of mice, with particular regard to carcinogenesis

The antifertility drug, Enovid, was tested for possible carcinogenicity in female mice of 5 specially selected strains: BALB/c, C3H, C3HfB, A, and C57BI. Enovid was chosen for testing since it is one of the most widely used oral contraceptives. The 5 strains of mice provided maximum genetic variation in the test animals. The drug was fed at 3 dose levels: 5 mcg/gm, 10 mcg/gm and 20 mcg/gm of food. The lowest dose did not prevent reproduction. The 10 mcg dose prevented some females from reproducing. The 20 mcg dose prevented all females from reproducing. The strains of mice differed in their response to Enovid. Weight gain was reduced in all strains. Effect on life-span varied, partly because of the tumors. Cervical and vaginal lesions showed invasion of the epithelium into the stroma but was limited, with few exceptions to the BALB/c females. In the BALB/c strain these lesions occurred in controls as well, but showed more progression and a higher incidence with the highest dose of Enovid. None of these lesions appeared grossly as tumors and none had extended beyond the vaginal wall or metastasized. They were observed only on histologic sections. Neither ovarian nor mammary gland tumors were increased in any strain. In the C3H strain such tumors seemed to be inhibited. In the C3HfB strain there was some inhibition of hepatomas and in the BALB/c strain some inhibition of adrenocortical adenoma. Chromophobe adenomas of the hypophysis were significantly increased in old C57BI females treated with the highest dose of Enovid. Offspring of Enovid-treated females showed no abnormalities. Enovid increased the occurrence and may have advanced the progression of epithelial lesions of the cervix and vagina of old BALB/c females. A study of the lesions in untreated females of this strain might help the understanding of carcinoma in situ in women and possibly the appearance of adeno-carcinoma of the vagina of young women whose mothers had been treated with stilbestrol during the first trimester to maintain pregnancy. Other neoplasms in this strain were not increased by the Enovid therapy. In the C3H strain mammary tumors were reduced by the Enovid and those that did occur were found later than in controls. Results from experimental animals should be applied to humans with care. Such results are of greatest value in directing attention to certain areas for investigation.     

Lack of effectiveness of antiestrogens RU 39,411 or keoxifene in the prevention of estrogen-induced tumors in Syrian hamsters.

As part of a search for an effective and safe antiestrogen to be used as adjunct therapy in the treatment of breast cancer, we examined the potential of RU 39,411 and keoxifene to inhibit the incidence of estradiol-induced kidney tumors in Syrian hamsters. Groups of 10 hamsters were chronically treated with implants of either keoxifene, RU 39,411, estradiol plus keoxifene, or estradiol plus RU 39,411 for 8 months. Five hamsters received only estradiol and 5 control animals remained untreated. There was a 100% kidney tumor incidence in estradiol-treated hamsters, which was not statistically different from that in animals co-treated with estradiol plus keoxifene (3 of 4 hamsters with tumors) or estradiol plus RU 39,411 (7 of 8 hamsters with tumors). Rodents treated only with antiestrogen remained tumor free. In addition to kidney tumors, testicular cancer was also found in animals cotreated with either estradiol plus keoxifene (2 of 4 hamsters with tumors) or estradiol plus RU 39,411 (3 of 8 hamsters with tumors). Two animals of this latter group also developed liver tumors. Testicular or liver neoplasms were not observed in hamsters implanted only with estradiol or only with antiestrogen. The lack of inhibition of estrogen-induced carcinogenesis in hamsters by RU 39,411 or keoxifene suggests that these two antiestrogens are not as effective as previously tested substances in inhibiting the appearance of this cancer. However, their concentrations were sufficient to induce, in combination with estradiol, the development of testicular tumors in these hamsters.     

Origin of the medulloblastoma experimentally induced by human polyomavirus JC

The origin of the medulloblastoma induced by JC virus (JCV) in golden hamsters was investigated by the in situ hybridization method. After inoculation of JCV into newborn hamsters, a few migrating cells in the cerebellar molecular layer as well as several cells in the internal granular layer hybridized with an antisense mRNA probe of JCV T-antigen on the 10th day. The number of cells positive for this probe decreased on the 15th and 20th days. Moreover, an incipient medulloblastoma consisting of many cells positive for T-antigen mRNA was noticed in the cerebellar internal granular layer 30 days after inoculation. About 6 months post inoculation, 95% of the animals had succumbed to medulloblastoma. Therefore, the origin of the medulloblastoma seemed to be the cells in the cerebellar external granular layer that were infected by JCV, that migrated normally through the molecular layer to the internal granular layer, and that began to proliferate to become medulloblastoma. When 5-day-old hamsters were inoculated, a few cells positive for T-antigen mRNA were detected in the cerebellum within a month. In the long-term observation, the ratio of medulloblastoma induction decreased as the age of the animals at inoculation increased. These results support the idea that JCV infects and transforms the cells in the cerebellar external granular layer, because this layer appears only transiently in newborn hamsters.     

Interspecies-, species- and type-specific T antigenic determinants of human papovaviruses (JC and BK) and of Simian virus 40.

Immunofluorescence tests, absorption studies and quantitative analysis by a very sensitive 51Cr microcomplement fixation (CF) technique were used to define the degree of relatedness between the tumor (T) antigens induced by human papovaviruses, strain JC and BK, with simian virus 40(SV40) and mouse polyoma virus (PyV). Antisera against JCV, BKV, SV40 and PyV T were raised in tumor-bearing hamsters. The data obtained indicate that T antigens of JCV, BKV and SV40 possess various subspecificities which can be distinguished and looked upon as interspecies-, species- and type-specific antigenic determinants. It was found that JCV T and BKV T synthesized in transformed hamster cells share about the same amount (20%) of interspecies cross-reacting antigen with SV40 T from H-50 cell extracts (transformed hamster cells). Although hamster cells transformed by PyV showed definite PyV T reactivity, no cross-reactivity, at least with the sera used, was found with human papovavirus and SV40 T antigens. Furthermore, degree of heterogeneity was observed within the T antigen complex derived from different SV40-transformed cells.     

Induction of undifferentiated brain tumors in rats by a human polyomavirus (JC virus)

Newborn SD rats were inoculated intracranially with JC virus (Tokyo-1), a human polyomavirus, isolated from the autopsied brain of a patient with progressive multifocal leukoencephalopathy. Twenty-one to 61 weeks later, 20 of 27 rats developed tumors in the cerebrum, but not in the cerebellum. The undifferentiated neuroectodermal nature of the tumors was histologically, immunohistochemically and ultrastructurally confirmed.     

Effect of adeno-associated virus on cancer expression by herpesvirus-transformed hamster cells.

Infection of herpes simplex virus type-2-transformed hamster tumor cells with adeno-associated virus type 1 before inoculation into hamsters specifically delayed the appearance of palpable tumors and increased the survival time of the animals. The data indicated that a defective virus of humans can influence cancer expression by a virus-transformed cell.     

Biological characteristics of primary tumors induced by polyoma virus in hamsters

Eleven primary subcutaneous tumors induced by polyoma virus in hamsters were analyzed for morphology, growth capacity in vitro, transplantability and karyology. In most cases, cultures prepared from these tumors grew for a few generations and then degenerated with loss of a majority of cells; from the few surviving cells, a population emerged that was able to grow indefinitely in vitro. The initial explanted population differed from that which emerged from the degenerative phase in its susceptibility to the toxic activity of 3,4-benzo (a)pyrene and in its high MTD (median tumor dose) values (10⁵–10⁶ cells) when transplanted in adult hamsters. The MTD decreased to 10²–10³ cells with prolonged growth in vitro. Cultures derived from the transplanted tumors grew permanently in vitro and had low MTD values. All the tumors were aneuploid with a prevalent near-diploid mode and remained so even after prolonged growth in vitro. No common karyotypic changes were observed, but in 4/8 tumors there was a chromosome marker longer than the X. The long chromosome of the various lines differed in the arm ratios. The occurrence of monosomy and trisomy of pairs ≠1 and ≠21 was not random in 4/8 primary tumors. A comparison with previous studies of hamster embryo cells infected in vitro with polyoma virus indicates that there is a strong parallelism in the biological properties and progression toward neoplasia between cell populations infected in vivoor in vitro by the virus.     

Isozyme phenotypes of polyoma virus tumors in mice

Isozyme profiles for 32 enzyme systems were studied in tumors induced by two strains of polyoma virus (2PTA and LID1), in two conventional mouse strains (C3H/BiDa and NIH), and in athymic (nude) mice of two genetic backgrounds (C3H/Hes nu/nu and NIH nu/nu). Tumors studied were: primary and transplant passages of salivary gland tumors (127); primary thymic epithelial tumors (12); primary subcutaneous sarcomas (6); primary hair follicle tumors (5); primary and transplant passages of mammary tumors (18); primary ameloblastomas (3); and primary renal medullary sarcomas (3). Regardless of mouse strain or virus strain, the isozyme arrays were highly constant and unique for each tumor histotype with the exception of salivary and mammary tumors, which shared a single profile differing from that of each of the other histotype-associated profiles. Other tumor types could be distinguished from each other and from the salivary-mammary tumor pair by as few as five isozymes: glycerol-3-phosphate dehydrogenase; glyceraldehydephosphate dehydrogenase; lactate dehydrogenase; sorbitol dehydrogenase; and alkaline phosphatase. Twelve nonpolyoma mammary tumors and their passages from mouse mammary tumor virus-expressed C3H/Hes nu/+ mice were analyzed for the same enzymes; variations in activity and isozyme profiles were found for ten enzyme systems. Three spontaneous salivary myoepitheliomas in BALB/c mice were also analyzed; two different lactate dehydrogenase profiles were observed, and all three tumors lacked the placental alkaline phosphatase present in polyoma virus-induced salivary tumors. Uniformity of isozyme phenotype may be characteristic of DNA virus transformation of cells in a particular differentiative state. This uniformity does not appear to occur in mouse mammary tumor virus-associated tumors, spontaneous tumors, and, according to the literature, chemically induced tumors.     

Mammary tumors, plaques, and hyperplastic alveolar nodules in various combinations of mouse inbred strains and the different lines of the mammary tumor virus

The following strains have been bred and developed for mammary tumor studies in this laboratory; C3H, C3HfB, C3HfDD, C3H-A^vy, C3H-A^vyfB, DD, DDfB, DDfC3H, BALB/c, BALB/cfDD, BALB/cfC3H, A (High), A (Low), AfB, and C57BL. Data are presented on mammary tumor incidence and age, occurrence of plaques, and occurrence of hyperplastic nodules. C3H-A^vy is our highest mammary tumor strain, whereas C57BL is the most resistant. A complete range of mammary tumor incidences and average ages depending upon the strain of mouse and line of virus is found in the other strains. The occurrence of mammary tumors in strains fostered on C57BL ranges from an incidence of 90% at an average age of 15 months in C3H-A^vyfB, although there is no evidence of any milk-transmitted virus, to 1.3% at 16.5 months in AfB. We continue to find an incidence of 20% at 17 months in BALB/c although we have no conclusive evidence of the virus. C3H virus has a greater potency than DD virus. Plaques are the result of DD or BALB/c genotype and DD virus but not of C3H or A genotype or of C3H or A virus. Hyperplastic nodules appear in all strains that develop mammary tumors, the age at which they appear depending upon the mouse strain and line of virus. These data form the foundation for other studies with these strains. The strains are now available to other investigators.     

Base-line data in a carcinogen-susceptible first-generation hybrid strain of Syrian golden hamsters: F1D Alexander

Base-line data in over 600 control animals of both sexes of the first-generation hybrid BIO F1D Alexander strain of Syrian golden hamsters are presented. They involve mortality, body weights, spontaneous tumor incidence, and incidence of nonneoplastic lesions. The results confirmed previously published data on smaller numbers of animals of this hamster strain. Spontaneous tumors with an incidence of more than 2% were limited to lymphomas (less than or equal to 6%), adrenocortical carcinomas (less than 8%), adrenal adenomas (9-14% in males; 3.5% in females), islet cell adenomas (less than 6%), and follicular adenomas of the thyroid gland (3.5% in females only). This low incidence of spontaneous tumors and the high survival rate (compared to those of hamsters from other sources), together with the previously established high susceptibility to tumor induction by carcinogen administration, render the F1D Alexander hamster an excellent animal model for lifetime carcinogenesis bioassays.     

Comparative carcinogenicity in F344 rats and Syrian golden hamsters of N′-nitrosonornicotine and N′-nitrosonornicotine-1-N-oxide

N′-Nitrosonornicotine (NNN) or N′-nitrosonornicotine-1-N-oxide (NNN-1-N-oxide), one of its metabolites, was added to the drinking water (0.012% for 36 weeks) of groups of male and female F344 rats or to the drinking water (0.016% for 31 weeks) of groups of male and female Syrian golden hamsters. All rats treated with NNN had died after 12 months but 50% of those treated with NNN-1-N-oxide survived for 22 months. NNN induced esophageal tumors in 23/24 rats and nasal cavity tumors in 21/24 rats. NNN-1-N-oxide induced esophageal tumors in 10/24 rats and nasal cavity tumors in 18/24 rats. There was no difference in survival rates among hamsters treated with either NNN or NNN-1-N-oxide. NNN induced tracheal tumors in 2/20 hamsters and nasal cavity tumors in 4/20 hamsters. NNN-1-N-oxide did not induce respiratory tract tumors in hamsters. These results demonstrate that NNN-1-N-oxide is less carcinogenic than NNN in F344 rats and Syrian golden hamsters.     

Serum antibodies to JC virus, BK virus, simian virus 40, and the risk of incident adult astrocytic brain tumors.

Genomic sequences of the human polyomaviruses, JC virus (JCV) and BK virus (BKV), and simian virus 40 (SV40) have been reported from several types of human brain tumors, but there have been no population-based seroepidemiologic studies to evaluate the association between polyomavirus infection and brain tumors. We conducted a case-control study, nested within a prospective cohort, to investigate the association between antibodies to JCV, BKV, and SV40, as measured in serum collected 1-22 years before diagnosis and incident primary malignant brain tumors. Brain tumor cases (n = 44) and age-, gender-, and race-matched controls (n = 88) were identified from participants of two specimen banks in Washington County, Maryland. IgG antibodies to the capsid proteins of JCV and BKV were assessed using ELISAs. SV40-neutralizing antibodies were measured using plaque neutralization assays. Similar to the general population, the prevalence of JCV and BKV infection was high in our study population (77 and 85%, respectively). Antibodies to SV40 were less prevalent (11%). The odds ratio for subsequent brain tumor development was 1.46 [95% confidence interval (CI), 0.61-3.5] for JCV, 0.66 for BKV (95% CI, 0.22-1.95), and 1.00 for SV40 (95% CI, 0.30-3.32). Given the high prevalence of JCV and BKV infections and the millions who were potentially exposed to SV40 through contaminated polio vaccines, future studies should attempt to replicate these findings.     

EBV-positive diffuse large B-cell lymphoma in a human T-lymphotropic virus type 1 carrier

Primary lymphoma of the adrenal gland is a rare and highly aggressive disease, with only a few reports in the literature. The pathogenesis is unknown, but detection of Epstein Barr virus (EBV) genome sequences and gene expression in some cases of primary adrenal lymphomas suggested the virus might be a causative agent of the malignancy. While investigating the presence of genome sequences of oncogenic viruses in a large series of adrenal tumors, both EBV and JC polyomavirus (JCV) DNA sequences were detected in a diffuse large primary bilateral B-cell non-Hodgkin lymphoma of the adrenal gland, which was diagnosed only at postmortem examination in a 77 year-old woman with incidentally discovered adrenal masses and primary adrenal insufficiency. The presence of both EBV and JCV genome sequences suggests the relevance of EBV and JCV coinfection in the pathogenesis of this rare form of B-cell lymphoma.     

Effect of simian virus 40 subcutaneous tumors on circulating lipids and lipoproteins in the Syrian hamster.

Circulating lipid levels and lipoprotein patterns in the Syrian hamster were determined at various times after subcutaneous inoculation with simian virus 40 (SV40) strain F, strain A-2895, or Fortner melanoma tumor cells. SV40 F tumors induced a rapid triphasic elevation of serum total lipids through inhibition of prebeta lipoprotein catabolism. Alpha lipoprotein levels declined in proportion to tumor mass. Liver wet weight and total lipid content increased significantly, but a normal rate of 3H-glycerol incorporation into polyanion precipitable (prebeta) serum lipoprotein was maintained. Determination of serum endogenous lipase, lecithin:cholesterol acyltransferase (LCAT), and cholinesterase activities indicated that these enzymes were not primarily responsible for the tumor-induced hyperlipidemia. Tumor-bearing animals also had selectively increased rates of protein and lipid excretion into the urine, with no evidence of gross hepatocellular or kidney damage. Growth of SV40 A-2895 tumors in hamsters resulted in a large increase in the rate of prebeta lipoprotein synthesis and degradation. Circulating prebeta lipoprotein levels were elevated much later in these animals, subsequent to a marked decrease in LCAT activity. Quite different results were obtained with Fortner melanoma, even large tumors having only a moderate effect on serum total lipid levels and lipoprotein patterns in the Syrian hamster.     

Polyoncogenicity and insulinoma-inducing ability of BK Virus, a human Papovavirus, in Syrian golden hamsters.

Newborn hamsters were inoculated intracerebrally with a series of purified and concentrated BK virus samples originating from a single stock of Gardner's original strain. Most (60-100%) of the hamsters developed various tumors 3-9 months later. The frequent types of tumors were ventricular tumors (choroid plexus papillomas and ependymomas: 7-53%), malignant insulinomas (0-92%), and osteosarcomas (0-50%). The T-antigen was positive in 59 of 60 tumors tested, but the virus was rescued by the cell fusion method from only 1 of 11 cell lines derived from these tumors. The incidence of insulinomas varied greatly with the virus sample; the two samples that showed the highest incidence (47 and 92%) originated from one parental virus stock, and all the other samples with the lower incidences (0-9%) originated from another ancestral stock. These results suggest the presence of a BK virus mutant(s) differing in capacity to induce insulinoma. A functional insulinoma cell line was thus established.     

Carcinogenicity of diallylnitrosamine following intragastric administration to Syrian hamsters

Single and multiple intragastric doses of diallylnitrosamine [(DAN) CAS: 16338-97-9] administered to Syrian golden hamsters induced tumors, primarily of the respiratory tract, in which the nasal cavity epithelium was the preferred site. When compared to the effect of DAN after subcutaneous administration at equal doses, the incidence of respiratory tract tumors was lower but that of hepatic tumors was higher, suggesting partial metabolism of DAN in the liver. Comparative metabolic and mutagenesis studies in BD IX rats (which reportedly are refractory to the carcinogenic effects of DAN), in Wistar rats, and in Syrian hamsters showed that a greater proportion of orally administered DAN was exhaled by both rat strains (12-19%) than by hamsters (2-4%). The activity of the microsomal fraction of the hamster liver for metabolizing DAN to allyl alcohol was about 10 times higher than that in rats, whereas no significant species differences were found with the cytosolic fraction. Pretreatment of animals with phenobarbital (PB) or pregnenolone-16 alpha-carbonitrile (PCN) did not influence either microsomal or cytosolic enzyme activities in hamsters, whereas about a tenfold increase in enzyme activities was seen after pretreatment with PB in both rat strains and following PCN in Wistar rats. Moreover, in bacterial mutagenesis assays, hamster liver microsomes were twice as active as those in BD IX rats. The results are discussed in relation to the carcinogenicity of DAN in rats and hamsters.